Means and methods for the specific modulation of target genes in the cns and the eye and methods for their identification

Title: Means and methods for the specific modulation of target genes in the cns and the eye and methods for their identification.Abstract: Provided are methods for the treatment of disorders of the central nervous system (CNS) and the eye. In particular, use of compositions comprising a compound capable of modulating a target gene or gene product is described for the preparation of a pharmaceutical composition for the treatment of disorders of the CNS and/or the eye, wherein the composition is designed to be administered outside the blood-CNS and the blood-retina barriers. Furthermore, methods are provided for identifying and obtaining nucleic acid molecules encoding polypeptides involved in CNS disorders or of the eye, methods for diagnosing said disorders as well as transgenic animal deficient in the expression of target genes identified in accordance with the described method. In addition, methods of identifying and isolating drugs that are particularly useful for the treatment of disorders related to the CNS and/or the eye are disclosed. ...

The Patent Description & Claims data below is from USPTO Patent Application 20120277288, Means and methods for the specific modulation of target genes in the cns and the eye and methods for their identification.

FIELD OF THE INVENTION

The present invention relates to methods for the treatment of disorders of the central nervous system (CNS) and the eye. In particular, the present invention relates to the use of compositions comprising a compound capable of modulating a target gene or gene product for the preparation of a pharmaceutical composition for the treatment of disorders of the CNS and/or the eye, wherein the composition is designed to be administered outside the blood-CNS and the blood-retina barriers. The instant invention further relates to methods of identifying and isolating nucleic acid molecules encoding polypeptides involved in CNS disorders or of the eye, methods for diagnosing said disorders as well as to transgenic animals, wherein the expression of target genes identified in accordance with the method of the invention has been modulated. In addition, the present invention relates to methods of identifying and isolating drugs that are particularly useful for the treatment of disorders related to the CNS and/or the eye.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

BACKGROUND ART

A variety of approaches currently exist for delivering biologically active agents to the CNS and/or the eye. These include, among possible others, oral administration, intravenous-, intramuscular- and transcutaneous administration as well as intra-bulbous injection or application as eye-drops. If the drug is delivered into the systemic circulation, it is being carried to all internal organs and tissues and it has to pass through the blood-brain and/or blood retina barrier (in order to access the CNS and/or the inner parts of the eye). Obviously, all other organs are being exposed to the drug, which may lead to a high incidence of side effects, particularly when the drug exerts its effects on target genes or gene products, which are not specific for the disorder to be treated and/or the target cell or tissue.

Another strategy often employed in brain delivery is the use of invasive methods such as intraventricular infusion systems, intracerebral (polymeric) implants, transplantation of genetically engineered protein-secreting cells and cell implants. These methods are unfortunately only effective for drug delivery to the surface of the brain or to cells immediately adjacent to the depot or infusion site and can be used for example in the treatment of carcinomatous infiltration of the meninges. However, these methods have many limitations because effective drug concentrations in brain parenchyma cannot be achieved.

Like the human central nervous system the human eye is an organ characterized by high complexity and the coordinated functioning of numerous specific structures and tissues. Both are protected by barriers (tear secretion, enzymes, transport mechanisms, blood-retina and blood-CNS barrier) against harmful environmental influences. Like the blood-brain barrier, the blood-retina barrier also represents a physiological barrier for the uptake of medication by the inner part of the eye, and makes pharmacological therapy of ocular diseases very difficult indeed—if at all possible—at the present state of technology.

Medication currently available on the market for the treatment of disorders of the CNS including ophthalmological diseases is therefore almost exclusively available for treatment of clinical symptoms often associated with side effects due to the high doses necessary. A causal therapy of the CNS, and particularly of the back sections of the eye, was not possible apart from the injections. Furthermore, the current state of information on the complex molecular metabolic interrelationship underlying the etiology of retinal diseases of multi-factorial origin is only limited. Consequently, medicaments available on the market are suitable to treat the symptoms of such diseases only.

In view of the need of therapeutic means for the treatment of diseases related to CNS and/or the eye, the technical problem of the present invention is to provide means and methods for the identification and modulation of genes involved in disorders of the CNS and/or the eye. More specifically, the technical problem of present invention is to provide non-invasive methods for the controlled modulation of target genes and gene products in the mammalian CNS and/or eye while overcoming the blood-brain and/or blood retina barrier without injuring it.

The solution to said technical problem is achieved by providing the embodiments characterized in the claims, and described further below.

SUMMARY

The present invention is directed to a method for the treatment of a disorder of the central nervous system (CNS) and/or the eye comprising administering to a subject a composition comprising a compound capable of modulating a target gene or gene product in a therapeutically effective amount, wherein said composition is administered outside the blood-brain and/or the blood-retina barriers. In particular, said composition can comprise one or more double-stranded oligoribonucleotides (dsRNA), which mediate an RNA interference of the corresponding mRNA of one or more target genes.

In another aspect, the present invention is directed to a method of identifying and isolating a nucleic acid molecule encoding a polypeptide involved in a disorder of the CNS and/or the eye comprising the steps of
(a) culturing a cell, tissue or non-human animal under stress conditions which lead to simulation of a pathological condition related to a CNS or eye disorder;
(b) isolating nucleic acids and/or proteins from a sample of said cell, tissue or animal;
(c) comparing the expression or activity profile of at least one of said nucleic acids and/or proteins with that of a corresponding non-treated cell, tissue or animal, and/or with that of a cell, tissue or animal, which has been treated under different stress conditions;
(d) determining at least one nucleic acid and/or protein which is differentially expressed, whereby a change of expression or of the active amount of said at least one nucleic acid or activity of at least one of said proteins or an altered localization of the protein is indicative for its role in a disorder of the CNS or eye.

The present invention also relates to nucleic acid molecules obtainable by the method described above, particularly if the encoded polypeptide is involved in angiogenesis and/or neovascularization and/or retinal disorder as well as to vectors comprising such nucleic acid molecules and host cells comprising said vector.

The present invention is also directed to a method for the production of a polypeptide capable of inducing a responsive change in a phenotype comprising culturing said host cell under conditions allowing the expression of the polypeptide and recovering the produced polypeptide from the culture as well as to polypeptides obtainable by said method or encoded by the nucleic acid molecules mentioned above.

Furthermore, the present invention relates to an antibody specifically recognizing such a polypeptide and pharmaceutical and/or diagnostic compositions comprising such an antibody or any one of the above described nucleic acid molecules, nucleic acid molecules which are complementary to such a nucleic acid molecules, vectors, host cells, and/or polypeptides, and optionally a pharmaceutically acceptable carrier and suitable means for detection, respectively.

In addition, the present invention is directed to methods for treating a disorder of the CNS and/or the eye comprising administering to the subject said pharmaceutical compositions in an effective dose,

Furthermore, the present invention relates to a method for detecting expression of a gene involved in a disorder of the CNS and/or eye comprising:
(a) obtaining mRNA from a cell;
(b) incubating the mRNA so obtained with a probe comprising a nucleic acid molecule described above or a fragment thereof under hybridizing conditions; and
(c) detecting the presence of mRNA hybridized to the probe; or
(a) obtaining a cell sample from the subject;
(b) contacting the cell sample so obtained with an antibody described above; and
(c) detecting the presence of the antibody bound to the protein encoded by said gene.

The invention furthermore is directed to a method for diagnosing in a subject said disorder or a predisposition to such disorder which comprises:
(a) isolating DNA from patient suffering from the disorder;
(b) digesting the isolated DNA of step (a) with at least one restriction enzyme;
(c) electrophoretically separating the resulting DNA fragments on a sizing gel;
(d) incubating the resulting gel with a probe comprising a nucleic acid molecule of the invention or a fragment thereof labelled with a detectable marker;
(e) detecting labelled bands on a gel which have hybridized to the probe as defined to create a band pattern specific to the DNA of patients of the disorder;
(f) preparing subject's DNA by steps (a) to (e) to produce detectable labeled bands on a gel; and
(g) comparing the band pattern specific to the DNA of patients of the disorder of step (e) and the subject's DNA of step (f) to determine whether the patterns are the same or different and to diagnose thereby the disorder or a predisposition to the disorder, if the patterns are the same; or
(a) analyzing a sample of nucleic acids of a subject by means of a diagnostic chip, primer extension, single nucleotide polymorphisms or sequencing comprising a nucleic acid molecule as described above; and
(b) comparing the result with that of a sample obtained from a patient suffering from the disorder;
wherein the identity of expression profil and/or nucleotide sequence is indicative for the disorder.

In further embodiment, the present invention relates to a method of determining whether a test substance has an effect on a nucleic acid molecule or polypeptide involved in a CNS or eye disorder comprising the steps:
(a) contacting a cell which expresses the target gene or gene product identified and isolated in accordance with the above described method with a compound to be screened; and
(b) determining if the compound modulates the expression or the activity of said target gene or gene product.

In a further aspect, the present invention relates to a drug or prodrug for the treatment of a disorder as defined above comprising:
(a) synthezising a test substance or a collection of test substances;
(b) subjecting said the test substance or collection of test substances to the screening method of the invention; and
(c) producing a compound identified as a modulator of a target gene or gene product or a derivative thereof.

In addition, the present invention is directed to a transgenic non-human animal which displays an aberrant expression or activity of the target gene or gene product defined above and to its use for a process in drug discovery for the treatment of said disorder.

DETAILED DESCRIPTION

The present invention relates to the use of a compound capable of modulating a target gene or gene product for the preparation of a pharmaceutical composition for the treatment of a disorder of the central nervous system (CNS) and/or the eye, wherein said composition is designed to be applied outside the blood-CNS and/or blood-retina barriers.

In one aspect, the present invention is based on the surprising finding that the blood-retina barrier could be overcome by the administration of compounds not considered to be capable of doing so in the therapy of ocular diseases by specific modulation of protein function in the tissues of the eye. Due to the functional similarity of the blood-retina barrier to the blood-brain barrier, providing an improved method to overcome the blood-retina barrier with the aim to treat a given eye disease is expected to be suitable for the treatment of CNS disorders, too.

Hence, in accordance with the present invention the compositions comprising a compound capable of modulating a target gene or gene product in the CNS or the eye are preferably designed to be administered without any substantial, i.e. substantially effective amount of delivery-enhancing agents facilitating passage of compounds through the blood-brain barrier and/or without the necessity of applying invasive methods and devices; see, e.g., those compounds, methods and devices described in US2002183683 and WO03/000018. However, for some embodiments, which represent independent aspects of the invention, such as the use of compounds mediating RNA interference, the use of such methods and compounds may be encompassed for the enhanced and controlled delivery of a compound capable of modulating a target gene or gene product into the mammalian CNS and/or eye while circumventing the blood-brain and blood-retina barriers.

Those later embodiments are based, inter alia, on the provision of novel methods that overcome the difficulty of the application of conventional experimental strategies for the identification of genes, which cause CNS disorders and/or eye diseases, and their validation as targets for diagnosis and for pharmacological intervention strategies. This applies especially for AMD, since the symptoms of this disorder appear only late, generally in the 7th decade of life. The current state of knowledge regarding the pathological metabolic interrelationships is not sufficient for the medical treatment of most CNS and eye diseases. Suitable animal or cell culture models are not available for such diseases, due to the complexity of the disease patterns and the lack of appropriate strategies for simple intervention and manipulation in the CNS and at the eye.

Hence, in one important aspect, the present invention relates to a cell, tissue and animal model based assay for the identification and isolation of target genes and gene products involved in disorders of the CNS and/or the eye and their use as targets for therapeutic intervention and/or diagnosis of such disorders.

The compositions of the invention may be administered locally or systemically e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer\'s dextrose, dextrose and sodium chloride, lactated Ringer\'s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer\'s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.

In accordance with the present invention the pharmaceutical compositions are administered to a subject in an effective dose of between about 0.1 μg to about 10 mg units/day and/or units/kg body weight; see also infra. Furthermore, the appropriate dosage regimen can be determined according to Example 21.

Some of these clinical phenotypes are characterized by a pathological de novo generation of blood vessels, which is called neoangiogenesis or neovascularization. Starting from the choriokapillaris, the growth of new blood vessels into the inner eye then leads to an increasing degeneration of photoreceptor cells in the affected areas of the human retina. In the field of opthalmology, one can distinguish between two forms of neovascularization: subretinal (choroidal=CNV) neovascularization and retinal neovascularization. Subretinal neovascularization, which is also called subfoveal neovascularization, is associated with degenerative disorders like Makular degeneration and characterized by loss of visual acuity and metamorphopsy. On the other hand, retinal neovascularization, vitreous body or Iris neovascularization is associated with ischemic processes (e.g. retinal vasculitis and diabetic retinopathy). Furthermore, neoangiogenesis is an important pathomechanism in different, non ophthalmological disease patterns such as tumor growth, arthritis and diabetic nephropathy. Therefore, in a preferred embodiment of the methods and uses of the present invention said disorder to be treated is related to angiogenesis and/or neovascularization and particularly preferred to the retinal pigment epithelium (RPE), neurosensory retina and/or choriodea. Most preferred, the disorder is wet age-related macular degeneration (AMD) or diabetic retinopathy.

The following description deals with AMD as example for a complex eye disease with a genetic component. Considering the wet form of AMD, it also serves as an example for a disease pattern, which is characterized by a distinct neovascularization. The example shall illustrate the associated technical problems with reference to the study of molecular causes and the development of diagnostic and pharmacological intervention strategies. AMD, which can be thought as a sub-type of retinal degeneration, is the most common cause of visual morbidity in the developed world with a prevalence increasing from 9% in persons over 52 years to more than 25% in persons over the age of 75 (Paetkau et al. 1978, Leibowitz et al. 1980, Banks and Hutton 1981, Ghafour et al. 1983, Hyman 1987, Hyman et al. 1983, Grey et al. 1989, Yap and Weatherill 1989, Heiba et al. 1994).

An early stage in the evolution of AMD pathology is accompagnied by an increasing accumulation of yellowish lipofuscin-like particles within the retinal pigment epithelium (RPE; Feeney 1978). It is thought that these particles represent remnants of undigested phagocytosed photoreceptor outer segment membranes which, in the normal process, are excreted basally through Bruch\'s membrane into the choriocapillaris. Over time, accumulation of lipofuscin-like particles affect Bruch\'s membrane and lead to its progressive destruction (Hogan and Alvarado 1967, Sarks 1976, Feeney-Burns and Ellersieck 1985, Pauleikhoff et al. 1990). The deposits in the RPE and Bruch\'s membrane consists largely of lipids although their exact composition may vary between individuals with some deposits revealing more polar phospholipids while others contain predominantly apolar neutral lipids.

These individual differences in drusen composition are thought to be the basis for the clinical heterogeneity in AMD (Green et al. 1985). While some patients present with an ingrowth of vessels from the choriocapillaris through Bruch\'s membrane (neovascularization) (Bressler et al. 1982), others show pigment epithelial detachment due to excudation underneath the RPE (Gass 1967, Green et al. 1985), and a third group of patients experiences a slow decrease of visual loss due to atrophic changes in the RPE and the overlying sensory neuroretina (Maguire and Vine 1986).

Although much less common the excudative/neovascular form of AMD accounts for more than 80% of blindness with a visual acuity of <20/200 (Bressler et al. 2002). In contrast to the above described “dry” form of AMD, the exudative “wet” AMD is associated with a choroidal neovascularization (CNV), leading to blindness and, thus, to a loss of life quality (followed by psychic disorders, increased risk of injury etc; Bressler et al. 2002). There is a high risk of developing (>40%) CNV in the second eye within 5 years of the development of CNV-AMD in the first eye (Bressler et al. 2002). Neovascular AMD is characterized by choroidal neovascular lesions. These lesions develop when abnormal blood vessels from the choroid grow and proliferate through breaks in the Bruch membrane to beneath the retinal pigment epithelium (Bressler et al. 2002, Campochiaro et al. 1999). The abnormal leakage from these vessels can result in hemorrhage or detachment of the retinal pigment epithelium or the neurosensory retina (which overlies the retinal pigment epithelium). Accompanying scar formation can replace retinal tissue and result in permanent vision loss.

AMD is a complex disease caused by exogenous as well as endogenous factors (Meyers and Zachary 1988; Seddon et al. 1997). In addition to environmental factors, several personal risk factors such as hypermetropia, light skin and iris colour, elevated serum cholesterol levels, hypertension or cigarette smoking have been suggested (Hyman et al. 1983, Klein et al. 1993, Sperduto and Hiller 1986, The Eye Disease Case-Control Study Group 1992, Bressler and Bressler 1995). A genetic component for AMD has been documented by several groups (Gass 1973, Piguet et al. 1993, Silvestri et al. 1994) and has lead to thehypothesis that the disease may be triggered by environmental/individual factors in those persons who are genetically predisposed. The number of genes which, when mutated, can confer susceptibility to AMD is not known but may be numerous.

The late onset of symptoms generally in the 7th decade of life as well as the clinical and likely genetic heterogeneity make it difficult to apply conventional approaches for the identification of genes predisposing to AMD. Due to the complexity of the clinical phenotype, it may be assumed that the number of genes is large, which, when mutated contribute to AMD susceptibility.

With recent physical approaches for the treatment of AMD such as laser photocoagulation, photo dynamic therapy (using verteprofin, trade name Visudyne®, Novartis), irradiation or surgical therapies, success was only achieved with a moderate percentage of the patients (Bressler et al. 2002, Yuzawa et al. 2001).

Hence, the methods, uses and compositions of the present invention described herein represent an important improvement and alternative therapeutic intervention for the treatment of this particular disease as well as of others. For those embodiments the pharmaceutical compositions are preferably designed to be effective in (and applied to) the posterior segment of the eye, preferably in a form designed to be applied outside the retinal region of the blood-retina barrier.

In one embodiment of the invention said compound is an inhibitor/antagonist of said target gene or gene product and preferably inhibits the expression of a gene or the activity of a gene product involved in angiogenesis and/or neovascularization; see supra.

The term “antagonist/inhibitor” in accordance with the present invention includes chemical agents that modulate the action of a gene or the activity of a gene product either through altering its enzymatic activity or through modulation of expression, e.g., by affecting transcription or translation. In some cases the antagonist/inhibitor may also be a substrate of a a gene product involved in the disorder or a ligand binding molecule.

The term “inhibitor” includes both substances which reduce the activity of the polypeptide and those which nullify it altogether.

An “antagonist” that modulates the activity of the gene product and causes for example a response in a cell based assay described below, refers to a compound that alters directly or indirectly the activity the gene product or the amount of active product. The effect of an antagonist may be observed as a blocking of agonist-induced activation of a target gene. Antagonists include competitive as well as non-competitive antagonists. A competitive antagonist (or competitive blocker) interacts with or near the site specific for agonist binding. A non-competitive antagonist or blocker inactivates the function of the gene product by interacting with a site other than the agonist interaction site. Preferably, the antagonist/inhibitor is small chemical agent which directly interacts with the target gene product involved in the disorder, preferably with a gene product involved in angiogenesis and/or neovascularization. Therefore, there will preferably be a direct relationship between the molar amount of compound required to inhibit or stimulate the target gene activity and the molar amount of gene product present or lacking in the cell. The compounds can be derived from a polypeptide, an anti-polypeptide antibody, an RNA molecule encoding (part of) a polypeptide or its antisense sequence, a transcription regulator, a ligand binding molecule, a polypeptide substrate or a known agonist/activator or antagonist/inhibitor.

In a preferred embodiment of the present invention said antagonist is based on nucleic acids, for example a ribozyme, antisense or sense nucleic acid molecules to said gene or gene or dsRNA molecules which are capable of mediating RNA interference. Methods and computer programs for the preparation rational selection of for example antisense oligonucleotide sequences are described in the prior art; see for example Smith, Eur. J. Pharm. Sci. 11 (2000), 191-198; Toschi, Methods 22 (2000), 261-269; Sohail, Adv. Drug Deliv. Rev. 44 (2000), 23-34; Moulton, J. Comput. Biol. 7 (2000), 277-292. These procedures comprise how to find optimal hybridization sites, and secondly on how to select sequences that bind to for example mRNAs overexpressed in a CNS or eye disorder. These methods can include the more empirical testing of large numbers of mRNA complementary sequences to the more systematic techniques, i.e. RNase H mapping, use of combinatorial arrays and prediction of secondary structure of mRNA by computational methods. Structures that bind to structured RNA, i.e. aptastructures and tethered oligonucleotide probes, and foldback triplex-forming oligonucleotides can also be employed for the purpose of the present invention. Relating to selection of antisense sequences by aid of computational analysis, valuable www addresses are given below.

In a particularly preferred embodiment of the present invention said antagonist/inhibitor substantially consists of ribonucleotides which preferably contain a portion of double-stranded oligoribonucleotides (dsRNA). Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites is described for example in Amarzguioui, Nucleic Acids Res. 28 (2000), 4113-4124. Minimising the secondary structure of DNA targets by incorporation of a modified deoxynucleoside: implications for nucleic acid analysis by hybridisation is described in Nguyen, Nucleic Acids Res. 28 (2000), 3904-3909.

dsRNA between 21 and 23 nucleotides in length is preferred. The dsRNA molecule can also contain a terminal 3′-hydroxyl group and may represent an analogue of naturally occurring RNA, differing from the nucleotide sequence of said gene or gene product by addition, deletion, substitution or modification of one or more nucleotides. General processes of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell comprising RNA with double-stranded structure, i.e. dsRNA or RNAi are known to the person skilled in the art and are described, for in WO99/32619, WO01/68836, WO01/77350, WO00/44895, WO02/055692 and WO02/055693, the disclosure content of which is hereby incorporated by reference.

The target mRNA of said dsRNA is preferably encoded by gene or a cDNA obtained in accordance with the method of the present invention described below. In one embodiment the target nucleotide sequence encodes an amino acid sequence of SEQ ID NO: 2 or 4 and/or comprises a nucleotide sequence of SEQ ID NO: 1 or 3.

In one embodiment of the invention the compound to be used in the compositions is a nucleic acid molecule or encoded by a nucleic acid molecule and is designed to be expressed in cells of the CNS and/or eye. For those embodiments gene therapy intervention is envisaged; see also infra.

In a preferred embodiment of the methods and uses of the present invention the composition is in a form designed to be introduced into the cells or tissue of the CNS or eye by a suitable carrier, characterized by the application occurring outside the blood-CNS and/or blood-retina barriers, for instance as eye drops. It can also be administered systemically, iontophoretically or by retrobulbar injection.

Iontophoresis has been defined as the active introduction of ionised molecules into tissues by means of an electric current. The technique has been used to enhance drug delivery into tissues underlying the donor electrode (e.g. skin) as well as to the general blood circulation, thus providing systemic delivery of a drug to the entire body. Iontophoresis devices require at least two electrodes, both being in electrical contact with some portion of a biological membrane surface of the body. One electrode commonly referred to as the “donor” or “active” electrode, is the electrode from which the biologically active substance, such as a drug or prodrug, is delivered into the body. Another electrode having an opposite polarity functions to complete the electric circuit between the body and the electrical power source. This electrode is commonly referred to as the “receptor” or “passive” electrode. During iontophoresis, an electrical potential is applied over the electrodes, in order to create an electrical current to pass through the drug solution and the adjacent tissue. Iontophoresis has been described for the treatment of blood-vessel related disorders (e.g. restenosis), bladder, uterus, urethra and prostate disorders. U.S. Pat. Nos. 6,219,557; 5,588,961; 5,843016; 5,486,160; 5,222,936; 5,232,441; 5,401,239 and 5,728,068 disclose different types of iontophoresis catheters for insertion into hollow, tubular organs (bladder, urethra and prostate) or into blood vessels. US 2002183683 suggests the method for delivery of active substances into the CNS.

Numerous active, often specifically expressed genes are required to perform and control the processes in the cells of the CNS and the retina and the metabolic exchanges across the blood-CNS and blood-retina barrier. Specific genetic activity is also necessary for maintaining the structure and functional integrity of numerous components of these complex tissues. As a consequence, this unique and highly evolved system is especially susceptible to various genetic defects, thus leading to a wide range of disease phenotypes. While studying monogenetic disorders is relatively easy, provided the patients are members of a family sufficiently large enough to allow positional cloning, the identification of genes that contribute to multigeneic disorders or confer or susceptibility to a disease is far more difficult.

Hence, in another aspect the present invention relates to a method of identifying and isolating a nucleic acid molecule encoding a polypeptide involved in a disorder of the CNS and/or the eye comprising:
(a) culturing a cell, tissue or non-human animal under stress conditions which lead to simulation of a pathological condition related to a CNS or eye disorder;

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